Systemic Chemotherapy of Melanoma: NMR Studies of Lonidamine & N-Mustard Activity

描述(申请人提供)：黑色素瘤仍然是人类最致命的癌症之一，目前还没有有效的方法来治疗这种播散性疾病。我们建议开发一种有效的全身化疗方法，通过[肿瘤]选择性[细胞内]酸化来使黑色素瘤对N-芥子烷基化药物增敏。我们的策略是联合给予葡萄糖和单羧酸转运(MCT)抑制剂LONIDAME LND，以捕获肿瘤中糖酵解产生的乳酸，并通过阻止丙酮酸转运到线粒体来抑制氧化代谢。这一方法依赖于MCT是肿瘤细胞内pH(Phi)动态平衡的关键途径，这已在我们的黑色素瘤模型中得到证实。以前在高血糖条件下接受LND治疗会导致一些死亡。我们在这个2年的R01项目上获得资助，以开发一种安全、可重复性的方法，用于用LND酸化人DB1黑色素瘤移植瘤。LND 100 mg/kg ip。在没有葡萄糖的情况下，我们已经实现了将肿瘤酸化到phi为6.4？0.1的至少一个小时，在此期间肿瘤的NTP/PI比率下降了50%；没有死亡，没有显著的体重减轻，与这种治疗相关的毒性最小。[LND对正常组织细胞外pH(Phe)的影响很小，但将肿瘤质膜的pH梯度从治疗前的pH6.9、Phe 7.0或pH=~0修改为治疗后的Phi=6.4、Phe=6.85或pH=-0.45。与LND前相比，肿瘤中的稳态乳酸水平增加了3倍。]呼吸、心电图、电解质水平和血氧含量没有变化，而血液分析仍在进行中。骨骼肌的pH值和ATP/PI无明显变化，肝脏的pH值和ATP/PI仅有一过性小幅下降。马法兰(LPAM7.5 mg/kg i.v)+LND比LND和LPAM分别延迟生长10.5±0.5d、0.6±0.7d和1.4±0.1d，LPAM对酸化的反应增强归因于活性中间体(氮杂环丙烷离子)浓度的增加、谷胱甘肽-S-转移酶活性的降低和O6-烷基转移酶的酸抑制导致的DNA修复的减少。肿瘤体积减少了52.5%±12.5%，与估计的对数10细胞杀伤=0.3012=对数10(2.0)一致。这些初步研究为实施多剂量系统治疗人类黑色素瘤异种移植模型奠定了基础，为最终的临床试验做准备。这项更新建议的目的是：1)通过低水平的葡萄糖与LND共同给药，最大限度地提高肿瘤的酸化程度，同时仍然避免危及生命的毒性，并将这些研究扩展到更具糖酵解能力的DB8黑色素瘤人异种移植模型。2)由于其他N-芥子剂在酸性phi值下可能表现出类似的活性增强，但毒性可能较低，LPAM将在单剂量和多剂量给药下与环磷酰胺、氯苯丁胺和苯达莫司汀进行比较。3)评价磁共振波谱(MRS)、表观弥散常数(ADC)和T2的核磁共振氢谱(1HMRI)无创早期检测反应--乳酸和总胆碱的可能性。

DESCRIPTION (provided by applicant): Melanoma remains one of the deadliest of human cancers with no effective method for treating the disseminated disease. We propose to develop an effective method for systemic chemotherapy of melanoma by [tumor] selective [intracellular] acidification to sensitize melanomas to N-mustard alkylating agents. Our strategy is to coadminister glucose with the monocarboxylic acid transport (MCT) inhibitor lonidamine LND in order to trap lactic acid produced by glycolysis in the tumor and to inhibit oxidative metabolism by impeding pyruvate trans- port into mitochondria. This approach depends on the MCT being the key pathway for tumor intracellular pH (pHi) homeostasis, which has been confirmed in our melanoma models. Previous treatment with LND under hyperglycemic conditions led to some mortality. We were funded on this 2yr R01 to develop a safe reproducible method for acidification of human DB1 melanoma xenografts with LND. By administering LND 100 mg/kg i.p. without glucose, we have achieved acidification of the tumor to a pHi of 6.4¿0.1 for at least hr during which time the NTP/Pi ratio of the tumor decreased by 50%; there was no mortality, no significant loss of weight and minimal toxicity associated with this treatment. [LND induced minimal changes in extracellular pH (pHe) of normal tissue, but modified the pH gradient across the plasma membrane of the tumor from pretreatment values of pHi 6.9, pHe 7.0 or !pH=~0 to a post-treatment values of pHi=6.4, pHe=6.85 or !pH=-0.45. Steady state lactate in the tumor increased 3-fold relative to pre-LND levels.] Respiration, EKG, electrolyte levels and blood oxygenation did not change, while blood analysis is still in progress. The pHi and ATP/Pi of skeletal muscle did not change, and there was only a small transient decrease in pHi and ATP/Pi of the liver. Melphalan (LPAM 7.5 mg/kg i.v.)+ LND exhibited a growth delay of 10.5¿0.5 d vs. LND alone, 0.6¿0.7 d. and LPAM alone 1.4¿0.1 d. The [enhanced] response of LPAM to acidification is attributed to an increase in the concentration of the active intermediate (aziridinium ion), a decrease in GSH due to decreased activity of glutathione-S-transferase and a decrease in DNA repair resulting from acid inhibition of O6-alkyl-transferase. Tumor volume decreased by 52.5 ¿12.5%, consistent with the estimated log10 cell kill=0.3012 = log10(2.0). These preliminary studies set the stage for implementation of multidose systemic treatment of xenograft models of human melanoma in preparation for eventual clinical trials. The Aims of this renewal proposal are: 1) To maximize tumor acidification while still avoiding life threatening toxicity by coadministration of low levels of glucose with LND, and to also extend these studies to the more glycolytic DB8 melanoma human xenograft model. 2) Since other N-mustard agents might exhibit similar enhancements of activity at acidic pHi values but perhaps with lower toxicity, LPAM will be compared with cyclophosphamide, chlorambucil and bendamustine under single and multidose administration. 3) To evaluate potential 1H NMR markers for noninvasive early detection of response--lactate and total choline by MRS and apparent diffusion constant (ADC) and T2 measured by MRI.